Explaining deuterium-depleted water as a cancer therapy: a narrative review

Introduction

Deuterium (heavy hydrogen) plays a critical role in mitochondrial disease. It interferes with the synthesis of ATP by mitochondrial F1F0-ATP synthase (F-ATPase) and induces the release of high amounts of reactive oxygen species (ROS) (Olgun, 2007). Nicotinamide adenine dinucleotide (NADH) is a critical player in the maintenance of low deuterium in mitochondrial water. Since NADH dehydrogenase directly supplies protons to F-ATPase in mitochondrial respiratory complex I. The proton supplied by NADH is unlikely to be a deuteron, as explained later in this article. Maintenance of the NAD pool in the mitochondria is essential for mitochondrial health, and the ratio of NAD⁺ to NADH is tightly regulated (Stein & Imai, 2012; Wikström & Hummer, 2012).

Cancer is a metabolic disease with a drastically altered metabolic policy, supporting rampant proliferation. Through the Warburg effect (increased energy supply obtained through glycolysis), cancer cells suppress oxidative phosphorylation in their mitochondria, while producing large amounts of lactate derived from glycolysis and releasing it into the external environment (Warburg et al., 1927; Potter et al., 2016). By reducing the role of ATPase in energy supply, however, they underutilize their mitochondria for energy supply, protecting them from DNA damage (through increased production of ROS).

During growth, cancer cells induce well above normal activity of the glycolysis pathway. This produces increased amounts of lactate that is excreted into the tumor microenvironment, which usually becomes acidic. Cancer cells need to survive and proliferate under extracellular hypoxic conditions and often under glucose deprivation, and they have developed special adaptations for this (for review see Hu et al., 2017). In order to maintain their basic intracellular pH and survive during hypoxic conditions, however, cancer cells need to intensify proton extrusion (Zheng et al., 2020) and increase their alkaline [e.g. bicarbonate (HCO₃⁻)] intake, which they achieve by altered and increased ion channel/transporter operation, for example, SLC4A4, found in most cancers. Altered ion channels under hypoxic conditions have been shown experimentally to promote the progression of gastric cancer (Chen et al., 2021).

Cancer cell growth is suppressed in a medium containing half the normal deuterium concentration, whereas it is accelerated with double the amount, opposite to the effect of deuterium on normal cells (Zhang et al., 2020). This suggests that cancer cells may be able to sense the amount of deuterium in the medium, and, when it is elevated, they are triggered to proliferate through complex signaling mechanisms. An experiment involving nasopharyngeal carcinoma (NPC) cells demonstrated that deuterium-depleted water (DDW) with deuterium concentrations at 100, 75, and 50 ppm suppressed the growth and invasiveness of tumor cells and caused cell cycle arrest. By contrast, DDW promoted the growth of normal control cells (Wang et al., 2012). DDW extends the lifespan of cancer patients in multiple studies (Somlyai et al., 2016, 2023; Yaglova et al., 2023).

Immune cells swarm to the tumor microenvironment, but signaling processes often suppress their ability to attack the tumor (Tie et al., 2022). ‘Tumor immune escape’ is a well-established phenomenon, mediated by programmed death ligand 1 (PD-L1)/PD-1 signaling (Jiang et al., 2019). An optimal T-cell response, a hallmark of successful cancer immunotherapies, is essential for efficiently killing cancer cells (Waldman et al., 2020). Kovács et al. (2022) exposed mice to carcinogens, and then the animals were supplied with DDW at 30 ppm deuterium. DDW treatment prevented tumor development compared with control mice. It is conceivable that immune cells regain mitochondrial health due to DDW, which then enables their antitumor effectiveness.

Here, we investigate the effects of DDW on the metabolic policy of cancer cells. DDW launches a cascade in cancer cells that results in suppressed autophagy and commitment to apoptosis. The effects of DDW treatment on cancer cell cycle arrest are explained through metabolic dependence of cancer on autophagy to discard damaged organelles and continue proliferation. Autophagy is a critical internal recycling process that cancer cells use to survive (Alvarez-Meythaler et al., 2020).

Lactate and isomerases

Isomerases are enzymes that convert two isomers back and forth from one to the other, a seemingly futile reaction, but, importantly, this achieves deuterium scrubbing from the isomers. Phosphoglucose isomerase in spinach chloroplasts has a very strong deuterium kinetic isotope effect (KIE), which results in essentially stripping deuterium from C2 of glucose (Maloney et al., 2024). The first pathway in glucose metabolism in a human cell is anaerobic glycolysis, which takes place in the cytoplasm and yields pyruvate as the product. Glucose is first converted into two isomeric triose phosphates, and a triosephosphate isomerase converts them back and forth from one to the other while depleting deuterium on the affected carbon atom (Leadlay et al., 1976). The enzyme has a high deuterium KIE in both directions and any deuterium that was initially present in glyceraldehyde-3-phosphate (G3P) exchanges with hydrogen in the medium as an intermediary step in the reaction. The repeated cyclic activity of the isomerase eventually nearly completely scrubs deuterium from the carbon atom in G3P that ultimately delivers a proton to NAD⁺ via a dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase), in the next step in the pathway. Thus, the proton in the NADH molecule is highly unlikely to be deuterium.

Lactate is produced from pyruvate by reverse activity of the enzyme lactate dehydrogenase (LDH), which transfers a deuterium-depleted (deupleted) proton from NADH to pyruvate. Hence, lactate released from cancer cells carries a low-deuterium proton that becomes very valuable to the recipient cell, which can convert lactate back to pyruvate, supplying a deupleted proton to mitochondrial NAD^+, and, ultimately, to the ATPase pumps via NADH dehydrogenase.

Vacuolar ATPase and microenvironment proton deupletion

Vacuolar ATPase (V-ATPase) plays a critical role in cancer pathology, and it is considered an attractive therapeutic target in cancer (Whitton et al., 2018). The two main types of ATPases in biological organisms are F-ATPase and V-ATPase. F-ATPase in the mitochondria synthesizes ATP from ADP while reducing oxygen to metabolic water. V-ATPases have an opposite function. Protons are pumped across a membrane from a proton-sparse medium to a proton-dense medium, and this requires energy, which is supplied by converting ATP to ADP. When localized to the plasma membrane, they pump protons into the extracellular space; whereas, when at the membrane of the endosome/lysosome organelle, they can acidify the endosome to convert it to a lysosome (Perzov et al., 2001).

A study evaluating the deuterium KIE of yeast V-ATPase acting at the plasma membrane discovered that the extrusion of H⁺ ions was reduced by as much as 90% when the cells were suspended in heavy water. The authors wrote: ‘It thus appears that the binding site [in V-ATPase] for protons (or hydronium ions) to be transported does not accept deuterons (or deuteronium ions) with equal ease or perhaps not at all’ (Kotyk et al., 1990). Thus, yeast V-ATPase has an extremely high deuterium KIE. Human breast cancer cells with high metastatic potential overexpress V-ATPase at the plasma membrane, and they use it presumably to transfer deupleted protons into the extracellular space, thus enriching deuterium levels in the cell’s cytoplasm and decreasing deuterium levels extracellularly (Sennoune et al., 2004).

Vacuolar ATPase, acidification, lactate, and M2 macrophages

Most tumors have a slightly elevated internal pH (7.3–7.7) compared with a low external pH (5.6–7.0), where the polarity is opposite to that of normal cells. Lactate concentrations in the tumor microenvironment can be as high as 40 mmol. The low pH in the tumor microenvironment is caused predominantly not by lactate but by the activities of V-ATPase pumps. The abnormal overexpression and unusual subcellular locations of V-ATPases in tumor cells are associated with a poor prognosis in many types of cancer. Plasma-membrane V-ATPase promotes tumor cell invasion, metastasis, and drug resistance (Chen et al., 2022). The release of lactate and acidification of the neighboring microenvironment by tumor cells accompanies metastasis, angiogenesis, and cancer progression (Pérez-Tomás & Pérez-Guillén, 2020).

Lactate is a valuable nutrient to fuel the tricarboxylic acid (TCA) cycle in regulatory T cells and bone-marrow-derived macrophages. LDH expression was found to be essential for CD8⁺ T cells to promote their cytotoxic activity (Apostolova & Pearce, 2022). LDH results in the synthesis of NADH to support deupleted protons for the ATPase pumps. The lactate anion is well tolerated by CD8⁺ T cells in pH-neutral conditions (Barbieri et al., 2023). When CD8⁺ T cells were exposed in culture to a pH-neutral environment along with high lactate availability during the T-cell activation phase, the lactate promoted rapid expansion and an enhanced effector profile, increasing their cytotoxic potential (Barbieri et al., 2023). Subcutaneous administration of sodium lactate solution at pH 7.4 to tumor-bearing mice leads to improved immune function of CD8⁺ T cells, by inhibiting histone deacetylases and boosting stem-like properties, resulting in significant suppression of tumor growth. Therefore, it is the low pH of the tumor microenvironment that promotes tumor progression rather than the lactate itself (Feng et al., 2022).

Excess lactic acid in the tumor microenvironment, however, indicates immunosuppression (Caslin et al., 2021). Lactate facilitates the recruitment of immunosuppressive cells, including tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and myeloid-derived suppressor cells. TAMs, the most abundant immune cells in the tumor microenvironment, play a powerful role in reprogramming T cells toward an immunosuppressive state, as well as promoting tumor initiation and progression (Hayes et al., 2021). Macrophages can be characterized as being in either an M1 (proinflammatory) or M2 (anti-inflammatory) state. Lactic acid skews macrophages toward an M2 state, particularly under low external pH conditions (Noe et al., 2021).

Lactate can induce marked changes in gene expression, in part through histone lactylation (Zhang D et al., 2019). In response to a bacterial infection, M0 macrophages differentiate into proinflammatory M1 macrophages, with an associated increase in glycolysis. As time passes, lactate accumulates in the cell, and this leads to histone lactylation, which then induces a transformation into an M2 phenotype, in preparation for wound healing and resolution of the inflammatory state. In TAMs isolated from lung tumors and melanoma cultures, the overexpression of Arg1, an M2-associated gene, was linked to lactylated but not acetylated histones. Exogenous lactate (e.g. released by cancer cells) also increased histone lactylation levels in immune cells and induced Arg1 expression, along with other M2-associated genes (Zhang D et al., 2019).

M2 macrophages facilitate tumor metastasis and augment drug resistance (Wang et al., 2024). Acidic pH, independent from lactate, can promote the polarization of macrophages toward an M2 state in prostate cancer cells. This promotes tumor survival, including enhanced tumor cell proliferation, loss of macrophage cytotoxicity, and release of angiogenic factors (El-Kenawi et al., 2019). Tumor relapse after chemotherapy is a major clinical problem, often involving inoperable metastasis. M2 macrophages accumulate around blood vessels after chemotherapy, promoting tumor revascularization and relapse (Hughes et al., 2015). In glioblastoma cells, extracellular vesicles containing lactate dehydrogenase A are released by macrophages recruited to the tumor microenvironment, to promote tumor cell glycolysis, proliferation, and survival (Khan et al., 2024).

Both LDH and monocarboxylate transporter 1 (MCT1), the transporter that imports lactate into the cell, show deuterium KIEs that would further support low deuterium levels in the mitochondria (Grimshaw and Cleland, 1980; Geistlinger et al., 2023). MCT1 has a significant deuterium KIE, with the uptake of lactate (and protons/deuterons) slowed significantly in D₂O at p²H 6.8 compared with H₂O at pH 6.8 (Geistlinger et al., 2023). A study on the effects of deuterium on the activity of human LDH found a solvent isotope effect of 2.75 for D₂O and a kinetic isotope of 2.74 for NAD²H instead of NADH, at pH 4.3 (Wang et al., 2016).

Deuterium-depleted water therapy as a treatment for cancer

DDW exerts strong antitumor activity (Wang et al., 2012; Zhang et al., 2020). Determining exactly how DDW suppresses cancer growth, however, has been a challenging task. Human clinical studies identify a potent anticancer activity obtained by DDW treatment (Somlyai et al., 2016, 2023; Boros et al., 2021; Yaglova et al., 2023). Glioblastoma multiforme (GBM) is an aggressive brain tumor with a relatively short median survival time (MST), especially at later onsets of disease (Wu et al., 2021). Patients who had sustained surgical excision of a brain tumor consumed wholly DDW as a daily fluid, to reduce deuterium levels in their body. DDW intake was given as an adjuvant therapy to chemotherapy and radiation. DDW extended their MST from 14.6 months to 30 months. DDW was especially effective for female patients, whose MST was increased to 42 months. Moreover, patients starting DDW concurrently with radiation lived for 47 months on average (Somlyai et al., 2023).

Pancreatic cancer is an aggressive and invasive cancer (Zhao and Liu, 2020). DDW in combination with conventional chemotherapy in pancreatic cancer patients remarkably increased their MST from 6.36 months to 19.6 months. These authors also conducted in vitro experiments where pancreatic cancer cells were grown in DDW. The lower the deuterium concentration the more the pancreatic cancer cell proliferation was inhibited (Boros et al., 2021). Deuterium depletion also delayed progression and prolonged MST in prostate cancer patients. DDW intake amount correlated inversely with prostate serum antigen levels. Moreover, DDW treatment significantly prolonged the MST of patients who suffered from distant metastasis (Kovács et al., 2011). DDW treatment is a promising anticancer approach as an adjuvant to either chemotherapy and/or radiation therapy, since cancer cells are sensitized to die upon deuterium depletion. DDW is a nontoxic anticancer strategy that has proved its efficacy in animal and human clinical trials (Lu & Chen, 2024). A number of indicative subclinical and clinical findings for DDW use as anticancer treatment are summarized in Table 1.

The role of cell cycle arrest

Deuteration (the exchange of hydrogen with deuterium) of active biomolecule structures involved in mitochondrial metabolism has long been shown to affect the normal mechanisms of hydrolysis of ATP (Dorgan & Schuster, 1981). In the case of cancer, one of the hallmarks of antitumor activity is to increase the rate of autophagy and/or apoptosis of malignant cells at certain stages of oncogenesis (Mulcahy Levy & Thorburn, 2020).

Autophagy is a catabolic process that sequesters damaged organelles into compartments known as autophagosomes, where they are degraded and removed. Autophagy plays a complex role in cancer cells. The Warburg effect is sustained by autophagy activation. The response to activation of autophagy depends on the stage and microenvironmental conditions. Autophagy often promotes cancer progression and metastasis, and it can facilitate multiple drug resistance (Alvarez-Meythaler et al., 2020). Stimulation of autophagy can reduce the toxic effects of deuterium through the turnover of defective mitochondria and heavily deuterated macromolecules. Autophagic mechanisms must be working efficiently to discard defective highly deuterated organelles (Olgun, 2007). Thus, the effects of deuteration can be delayed or prevented by decreasing the intake of deuterium and/or increasing the turnover of organelles and macromolecules by stimulating autophagy.

The application of DDW promotes the senescence of breast cancer cells (Lajos et al., 2018). Moreover, experiments on colorectal cancer cells treated with DDW showed similar senescent phenomena, but also revealed a proapoptotic effect, suggesting DDW-related promotion of autophagic and cancer cell death mechanisms (Chira et al., 2018). Further experiments on breast cancer cells showed that DDW treatment had a direct growth inhibitory effect, due to a G1/S cell cycle arrest, enhancing the anticancer activity of fluorouracil (5FU) (Yavari & Kooshesh, 2019). Recent experiments showed that DDW induces a similar G1/S phase cell cycle arrest that causes the death of colorectal cancer cells (Haseli et al., 2023). Moreover, DDW inhibits the proliferation, migration, and invasion of NPC cells. Strikingly, by contrast, the growth of normal preosteoblast cells (mesenchymal progenitors of osteoblasts) was promoted in the presence of DDW (Wang et al., 2013).

Understanding the mechanism of tumor suppression by deuterium-depleted water

Zhang X et al. (2019) used a proteomics approach to reveal a disbalance between ROS production and ROS neutralization brought on by DDW exposure to cancer cells, leading to suppression of activity of several proteins that maintain tumor growth. Tumor cells grown in culture and exposed to DDW experienced 32% inhibition of proliferation. The authors sought to understand exactly how DDW arrests tumor growth, causes oxidative stress, and induces apoptosis in tumor cells. The regulatory protein p62, overexpressed in cancer cells, was highly oxidized in response to the treatment. Oxidation of p62, an autophagy receptor protein, promotes its oligomerization which in turn leads to increased autophagy, including autophagy (and clearance) of p62 itself (Carroll et al., 2018; Islam et al., 2018). Decreasing its level lowers proliferation rates (Zhang et al., 2023).

This proteomics analysis revealed several specific enzymes whose expression was significantly downregulated in response to DDW treatment. The most downregulated enzymes are listed in Table 2. Most of these enzymes carry out reactions that involve either NADH or NADPH. Downregulation of aldehyde dehydrogenase 4 A1 would impair the ability of the tumor cell to convert proline to glutamate, decreasing its supply of glutamate, which is an important alternative fuel in the TCA cycle in cancer cells (Yi et al., 2019). Glutamate deprivation in prostate cancer cells decreased proliferation, migration, and invasion, leading to apoptotic cell death (Koochekpour et al., 2012). Both ferredoxin reductase and hexose-6-phosphate dehydrogenase replenish NADPH, which is needed to maintain glutathione in its reduced state. In parallel, glutathione peroxidase downregulation suppresses the ability to detoxify H₂O₂ in the mitochondria via glutathione oxidation. Dihydrofolate reductase (DHFR) regenerates tetrahydrofolate (THF) from dihydrofolate (DHF), so its suppression leads to a reduced supply of THF, an essential coenzyme for de novo synthesis of both pyrimidine and purine nucleotides, needed for DNA synthesis in proliferating cells. DHFR inhibitors such as methotrexate are common antifolate drugs used in cancer therapy (Taylor, 1993).

Zhang X et al. (2019) reported that the most highly oxidized enzyme was hydroxymethylglutaryl CoA synthase 1 (HMGCS1), the first step in the mevalonate pathway. This pathway leads to the synthesis of cholesterol, which is essential for assembling the plasma membranes of proliferating cells. Its suppression lowers mitochondrial membrane potential and increases the release of proapoptotic factors (Tricarico et al., 2015). Depletion of isoprenoids, other mevalonate pathway products, leads to endoplasmic reticulum (ER) stress and apoptosis in liver cells (De Giorgi et al., 2020).

DDW may not be toxic to the tumor cells per se, but rather cancer cells are programmed to respond to low deuterium by suppressing proliferation and inducing apoptosis. DDW induces complex metabolic changes in tumor cells that interfere with proliferation and promote autophagy and apoptosis. Dietary choices can also influence mitochondrial function. A high-fat (ketogenic) diet is a low-deuterium diet (Somlyai et al., 2022). Pesticide exposure can induce mitochondrial dysfunction and increase oxidative stress (Sule et al., 2022). Riboflavin deficiency can interfere with the supply of NAD(P)H (Schramm et al., 2014). These ideas are summarized in Fig. 1.

Discussion

Many of the unusual aspects of cancer metabolic policies can be explained as a strategy to produce and release deupleted nutrients and deupleted protons into the external milieu while concentrating deuterium internally. By severely restricting the activity of the F-ATPase pumps, tumor cells protect their defective mitochondria from the primary damaging effects of deuterium. M2-like TAMs infiltrating the tumor microenvironment promote tumor growth, angiogenesis, and metastasis and suppress T-cell activation (Wang et al., 2024). Nutrients supplied by the tumor, however, can support mitochondrial and lysosomal healing of the immune cells, eventually enabling them to become activated and destroy the tumor.

Strongholds and limitations on how deuterium-depleted water treatment works against cancer

A promising view, as deduced from results of DDW use in human clinical trials in prostate, lung, and pancreatic cancer studies, is that (1) deuterium can be regarded as a nutritional agent, and (2) its depletion from the organism extends the MST of cancer patients. DDW can be regarded as a nontoxic and easily noninvasive supplementation approach in these studies. The problem of cost in introducing the DDW approach, however, remains to be evaluated. The Boros et al. (2021) study describes an easy manufacturing laboratory process to produce DDW for patients. In this study, however, the optimum D content in DDW to provide the highest anticancer effect remains to be evaluated in future studies. Furthermore, the relatedness of first appearing cancer and its complications (metastasis) and the relation of DDW to oncogenesis stages of cancer remains to be elucidated further.

We had hoped in this article to be able to determine exactly how deuterium depletion works to attenuate essential factors that enforce cancer cell growth, but the ultimate answer still eludes us. It is plausible, however, that cancer cells, upon DDW treatment, suffer from levels of oxidative stress that cannot be handled. It has been observed experimentally that DDW induces apoptosis in cancer cells whereas it supports survival in normal cells (Wang et al., 2012). Oxidation of HMGCS1, as reported by Zhang X et al. (2019), likely leads to apoptosis following ER stress, due to depletion of isoprenoids. HMGGCS1 suppression also depletes the supply of cholesterol needed to populate the membranes of cell clones. The list of downregulated proteins in cancer cells in response to DDW provided in Table 1 predicts a deficiency in glutamate synthesis and in the regeneration of NADPH from NADP⁺. NADPH is urgently needed for antioxidant support when ROS production is high. Therefore, suppression of these critical enzymes may lead to sufficient mitochondrial damage to provoke mitochondrial outer membrane permeabilization, the release of cytochrome C into the cytoplasm, caspase activation, and a programmed apoptotic response (Lopez & Tait, 2015). We still do not know exactly how DDW induces these observed effects on protein expression in cancer cells, but our conjecture is that it is through an ability for cancer cells to sense deuterium levels in the medium and react to a reduction in deuterium via a signaling response that leads to these reductions in expression of critical proteins that support cancer survival and proliferation.

Finally, we want to highlight a remarkable new study conducted by a group of Russian scientists who showed that DDW fed to male Wistar rats delayed or even reversed thymic involution, a hallmark of immune cell aging. This study showed that, after a short time period of suppression, thymopoiesis not only becomes restored, but the rate of producing differentiated T cells exceeds the values of the animals that were not fed DDW (Yaglova et al., 2023). The activation of T-cell proliferation and the increase in the numbers of progenitor cells suggest that DDW counteracts and reverses thymic involution. Thymic involution is responsible for cancer emergence, relapse, and decreased immune anticancer activity (Wang et al., 2020). Therefore, the possible enhancement of thymic function in patients consuming DDW can be another important factor for the anticancer effects and prolongation of survival, a topic that urgently needs further research.

Much more research is needed to identify immune cell functions that are enhanced by DDW treatment. The clinical applications of DDW are relatively easily applied in medical settings and can be considered to have an impact above and beyond the already ongoing conventional therapeutic regimes.

Conclusion

A causal factor in oncogenesis is excess deuterium in mitochondria. Cells whose mitochondria are sufficiently damaged by deuterium may transform into tumor cells, and this transformation is associated with a readjustment of metabolism to reduce the need for mitochondria-derived energy. Simultaneously, they engage in a program that results in further enrichment of cytoplasmic deuterium through the export of deupleted protons and the overproduction of lactate, a carrier of a deupleted proton derived from NADH, exported to the medium. Cancer cells support deuterium sequestration and release deupleted nutrients, primarily lactate, to support neighboring immune cells and distant organs. The lactate and acidic pH produced by cancer cells lead to cancer progression and immunosuppression of resident immune cells.

DDW has promising therapeutic value in treating cancer. DDW appears to suppress autophagy and interfere with the clearance of damaged mitochondria, and this may then lead to cell cycle arrest. The substantial accumulation of ROS in cancer cells during DDW treatment causes senescence that leads to apoptosis. Clinical evaluation is needed to discover DDW’s influence on autophagic-related death by apoptosis at different stages of oncogenesis in different cancer types.

A future challenge would be to identify (or, ideally, reduce) the cost of DDW application in clinical settings providing anticancer treatment. Moreover, although many hydrogen/deuterium exchange studies prognose for the intracellular effects; these are primarily on a theoretical level. Apart from the phenomenal reduction of proliferation rate and induction of cell cycle arrest, which macromolecules are being affected during depletion of deuterium remains to be identified. This will enlighten more clinical anticancer approaches in the future.

Acknowledgements

This research was funded in part by Quanta Computer, Inc., Taiwan, under grant number 695075.